Structure-guided engineering of an aromatic dioxygenase from Coniochaeta pulveracea for improved catalysis of lignin-related aromatic olefins
Lei Yu (South China University of Technology)
Yunjian Ma (South China University of Technology)
Chenhao Feng (South China University of Technology)
Frank Hollmann (TU Delft - Applied Sciences)
Ren Wei (Greifswald University)
Fanghua Wang (South China University of Technology)
Shengjie Guo (South China University of Technology)
Bin Wu (Shunde Polytechnic, South China University of Technology)
Yonghua Wang (South China University of Technology, Southern Marine Science and Engineering Guangdong Laboratory)
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Abstract
Aromatic dioxygenases (ADOs) catalyse the oxidative cleavage of CC bonds in aromatic olefins, producing valuable aldehydes or ketones. Due to their coenzyme independence, ADOs are attractive catalysts. However, the limited catalytic performance of natural ADOs restricts their broader practical application. Here, we determined the 2.2 Å crystal structure of an ADO from Coniochaeta pulveracea (CpuADO). Structure-guided mutagenesis targeting residues near the active site identified a mutant F349W, which exhibits enhanced catalytic efficiency (kcat/Km) toward sinapyl alcohol by about 1.41-fold compared to the wild-type enzyme. In parallel, AI-based computational screening identified a mutant W338D, which shows improved catalytic efficiency for several aromatic olefins, including 1.3-fold for coniferyl alcohol, 1.7-fold for 4-vinylguaiacol, and approximately 12-fold for isoeugenol. Molecular dynamics (MD) simulations revealed stabilised Fe2+–CC distances (approximately 5.5 Å in F349W and 4.7–6.2 Å in W338D) and reduced structural fluctuations, indicating improved substrate positioning. These findings provide a structure-based strategy for engineering ADOs with enhanced catalytic performance toward lignin-related aromatic olefins, allowing for more efficient lignin valorisation.